Our goal is to determine the role of the inflammasome in disease mediated by anthrax lethal toxin (LT). The inflammasome, a critical part of innate immunity, is activated by numerous pathogens relevant to biodefense, including Bacillus anthracis. Strikingly, LT-mediated cell killing is controlled by inflammasome activation. It is unclear, however, how LT triggers the inflammasome, and causes subsequent cytopathic and pathogenic effects. The murine B. anthracis model is very useful for studying inflammasome involvement in microbial cell killing, since the inflammasome component Nalp1b has been linked to LT sensitivity of murine macrophages. Mice are divided into strains that harbor macrophages that are either susceptible or resistant to LT killing. Susceptible macrophages express a dominant Nalp1b allele, which appears to confer LT sensitivity, while resistant macrophages express a recessive Nalp1b allele that has been linked to LT resistance. Over the last 4 years we have acquired many valuable tools and methods to address the role of the inflammasome and Nalp1b in cell killing mediated by the anthrax exotoxin LT. These agents include tagged Nalp1b alleles derived from susceptible and nonsusceptible murine strains, as well as cell lines lacking different components of the inflammasome. We are therefore in an excellent position to address longstanding questions in anthrax and other microbial diseases. Based on our observations and findings by others, we hypothesize that expression of specific Nalp1b alleles determines the susceptibility of hematopoietic cells to LT killing, and that activation of caspase-1 by Nalp1b is required for this process. Our goal is to understand the role of Nalp1b and caspase-1 activation in LT killing, and specifically how LT triggers Nalp1b stimulation and cytopathic effects. [unreadable] [unreadable] Here we will test whether transducing Nalp1b alleles derived from susceptible murine macrophages confer sensitivity to LT resistant macrophages. Chimeric Nalp1b constructs, generated by exchanging corresponding fragments of Nalp1b alleles derived from susceptible and nonsusceptible murine macrophages, will be used to identify Nalp1b determinants for LT responsiveness. To understand of Nalp1b activation, we will analyze the protein composition of the inflammasome complex in LT-treated and untreated macrophages. Nalp1b and inflammasome-binding proteins will be identified using immunoprecipitation and proteomics. The role of Nalp1b-binding proteins in inflammasome activation and cell killing will be tested. These studies will shed light on the role of the inflammasome in LT killing, and contribute to elucidating general mechanisms in microbial pathogenicity involving the inflammasome. [unreadable] [unreadable] We plan to study the role of the inflammasome in anthrax pathogenesis. We will determine how stimulation of the inflammasome, which is part of the immune response, could lead to cell death and morbidity in anthrax. [unreadable] [unreadable] [unreadable] [unreadable]